Recent Advances in the Preparation, Structure, and Biological Activities of β-Glucan from Ganoderma Species: A Review

Ganoderma has served as a valuable food supplement and medicinal ingredient with outstanding active compounds that are essential for human protection against chronic diseases. Modern pharmacology studies have proven that Ganoderma β-d-glucan exhibits versatile biological activities, such as immunomodulatory, antitumor, antioxidant, and antiviral properties, as well as gut microbiota regulation. As a promising polysaccharide, β-d-glucan is widely used in the prevention and treatment of various diseases. In recent years, the extraction, purification, structural characterization, and pharmacological activities of polysaccharides from the fruiting bodies, mycelia, spores, and fermentation broth of Ganoderma species have received wide attention from scholars globally. Unfortunately, comprehensive studies on the preparation, structure and bioactivity, toxicology, and utilization of β-d-glucans from Ganoderma species still need to be further explored, which may result in limitations in future sustainable industrial applications of β-d-glucans. Thus, this review summarizes the research progress in recent years on the physicochemical properties, structural characteristics, and bioactivity mechanisms of Ganoderma β-d-glucan, as well as its toxicological assessment and applications. This review is intended to provide a theoretical basis and reference for the development and application of β-d-glucan in the fields of pharmaceuticals, functional foods, and cosmetics.


Introduction
Ganoderma has been one of the most popular mushrooms, both for medicine and food, in Asia for more than 2000 years [1]. Currently, approximately 300 species of Ganoderma have been identified worldwide [2], which principally includes G. lucidum, G. applanatum, G. tsugae, G. boniense, G. atrum, and G. sinense, and these Ganoderma species have a wide range of health-promoting effects. Recently, consumers have become more health conscious, resulting in a growing demand for high-quality, nutritious, and health-promoting natural products. Ganoderma is known as a "herb that brings the dead back to life" and has been used for centuries as a medicine or nutritional supplement for the prevention, control, and treatment of chronic diseases [3], such as autoimmune diseases, cardiovascular diseases, diabetes, digestive diseases, and malignant tumors. As a natural medicinal mushroom, Ganoderma is abundant in a variety of physiologically bioactive components (more than 600 compounds), including polysaccharides, steroids, triterpenes, glycoproteins, sterols, peptides [4], etc. In particular, polysaccharides are the principal bioactive ingredients found in the fruiting bodies, mycelia, spores, and fermentation broth of Ganoderma species; thus, they are considered as an important candidate for functional food, medicine, and cosmetics [5,6].
Among these Ganoderma polysaccharides, β-D-glucan acts as the main active polysaccharide and has received increasing attention as a result of its versatile pharmacological activities over the last decades [7], such as its antitumor, anti-inflammatory, antioxidant, immunomodulatory properties. In addition, β-D-glucan exhibits diverse biological and health-promoting effects depending on its natural sources (e.g., yeast, mushroom, bacteria, seaweed, and cereal) [8,9], extraction and purification strategies [10], and structural characteristics [11]. Apart from its pharmacological and nutritional values, β-D-glucan is also used in the food industry due to its gel-forming and thickening effects. For example, the addition of β-D-glucan contributes to improving the texture and sensorial properties of chicken breast [12], sausage [13], and ice cream [14]. Furthermore, β-D-glucan is widely applied in cosmetics industries for its ability to maintain skin health [8,15]. Therefore, β-D-glucan might be regarded as a potential therapeutic agent for diseases, a healthy dairy food, and for use in skin care supplements.
Over the past two decades, the extraction and purification, structural features, bioactivities, and probable mechanisms of β-D-glucan obtained from diverse mushrooms have all been extensively studied by our team and other researchers; however, few reviews have generalized and summarized β-D-glucan from the Ganoderma species, which seriously limits the development and utilization of Ganoderma polysaccharides. This review aimed to systematically summarize the research advances in the physicochemical properties, structural characteristics, bioactivities, potential action mechanisms, safety assessment, and applications of β-D-glucan derived from Ganoderma species, to provide a scientific foundation for the thorough development and use of β-D-glucan from mushrooms.

Extraction and Purification
Currently, polysaccharides (especially β-D-glucan) are one of the major bioactive macromolecules components in Ganoderma species, and hot water (HW) and chemical solutions (alkali or acid-alkali) are the most commonly used for polysaccharides extraction [16,17]. Meanwhile, the yield of β-D-glucan from Ganoderma species using traditional extraction methods (especially HW extraction) may be influenced by some extraction parameters [18], such as the extraction time, extraction temperature, and water-to-raw material ratio. At present, several assisted extraction strategies, including physical methods (microwave, ultrasound, and pressure), biological methods (enzyme), and combinations of these methods (ultrasonic/microwave/enzyme-assisted extraction), have been developed to overcome some of the drawbacks of traditional extraction methods for mushroom polysaccharides [19,20]. Alzorqi et al. [21] showed that the total content of β-D-glucan extracted by ultrasonic-assisted extraction (UAE) from the fruiting bodies of G. lucidum was higher than that of enzyme-assisted extraction (EAE) or HW extraction. Smiderle et al. [22] found that the extraction of β-D-glucan from G. lucidum using microwave-assisted extraction (MAE) could reduce the extraction time compared with pressurized liquid extraction (PLE). It is worth noting that the above-mentioned extraction techniques help to increase the extraction yield of β-D-glucan from Ganoderma species and improve the pharmacological activities of β-D-glucan [23]. After extraction, the crude polysaccharides from Ganoderma species were separated and purified by a series of purifications process including deproteinization and decolorization to obtain homogeneous fractions. Subsequently, column chromatography (i.e., cellulose chromatography, ion-exchange chromatography, and gel-filtration chromatography) was commonly employed to further purify the crude polysaccharides [24,25]. Finally, the purified polysaccharides were concentrated, dialyzed, and lyophilized to facilitate the subsequent structural characterization. Of note, the high purity of β-D-glucan from Ganoderma species was usually precipitated with absolute ethanol several times. Based on the published literature, the representation of extraction and purification methods for β-D-glucan is summarized and displayed in Table 1 and Figure 1.

Biological Activities and Molecular Mechanisms of β-D-Glucan
Currently, Ganoderma species are used as traditional food and medicine in China and other Asian countries due to their health-promoting effects. In particular, β-D-glucans extracted and purified from Ganoderma species have been shown to bring beneficial effects to human health, including immunomodulation, antitumor, antioxidant, and anti-inflammation properties. Moreover, this review summarizes the biological activities of β-D-glucan in Table 2, and its action mechanisms are presented in Figures 3 and 4.

Biological Activities and Molecular Mechanisms of β-D-Glucan
Currently, Ganoderma species are used as traditional food and medicine in China and other Asian countries due to their health-promoting effects. In particular, β-D-glucans extracted and purified from Ganoderma species have been shown to bring beneficial effects to human health, including immunomodulation, antitumor, antioxidant, and antiinflammation properties. Moreover, this review summarizes the biological activities of β-D-glucan in Table 2, and its action mechanisms are presented in Figures 3 and 4. Mouse splenic B cell proliferation and NO production ↑; Levels of IL-1β, TNF-α, IL-10 and IL-12p40 ↑; [37] 9 Fruiting bodies of G. australe LPS-induced macrophage 0-2.5 µg/mL β-D-glucan for 48 h Level of IL-6 and phagocytic activity ↑; [36] 10

Immunomodulatory Activity
Numerous studies have shown that Ganoderma polysaccharides maintain "the stability of the body's internal environment" by enhancing the immune function of the host [67,68]. Of note, β-D-glucan mainly consists of a repeating structure of D-glucose units in Ganoderma species, which exhibits an immunomodulatory effect and regulates innate im-

Immunomodulatory Activity
Numerous studies have shown that Ganoderma polysaccharides maintain "the stability of the body's internal environment" by enhancing the immune function of the host [67,68]. Of note, β-D-glucan mainly consists of a repeating structure of D -glucose units in Ganoderma species, which exhibits an immunomodulatory effect and regulates innate immune functions [69]. Studies have also confirmed that β-D-glucan with a backbone of (1→3)-Glcp exhibits a significant immunomodulatory activity [70]. As an immunomodulator, β-D-glucan from Ganoderma species can alleviate the development and progression of immune-related diseases by the activation of immune cells (i.e., NK cells [60], dendritic cells [58], lymphocytes [55], and macrophages [71]). For instance, treatment with β-D-glucan from G. sinense promoted splenocyte B-cell proliferation and increased inflammatory cytokine secretion in mononuclear cells and DC, as well as enhancing the nitric oxide level in RAW264.7 cells [37]. A randomized clinical trial showed that the administration of β-D-glucan from Lingzhi or Reishi medicinal mushroom enhanced the count of peripheral blood total lymphocytes in asymptomatic children 3 to 5 years old for 7 weeks, and no serious side effects were observed [72]. Moreover, Ganoderma β-D-glucan contributes to the protection of spleen and thymus function and increases IgA levels in the serum of cyclophosphamide-induced immunosuppressive model mice [24]. Currently, published articles have indicated that Ganoderma β-D-glucan may boost the body's immune system against COVID-19 [73,74]. Mechanistically, β-D-glucan derived from Ganoderma species possesses an inhibitory effect on immunosuppressive diseases through pathogen-associated molecular patterns (PAMPs) (Figure 3). For example, β-D-glucan treatment significantly activated mitogen-activated protein kinases (MAPKs) and nuclear factor-κB (NF-κB) signaling pathways through binding with the pattern recognition receptors (i.e., dectin-1) to induce immune responses [75] and accelerating the secretion of cytokines (i.e., interferon-γ (IFN-γ), tumor necrosis factor-α (TNF-α), and interleukins) by immune cells [76,77]. Other studies found that dectin-1 can cooperate with other specific pattern recognition receptors (i.e., toll-like receptor 2 and complement receptor 3) of β-D-glucan can trigger innate immunity [78,79]. Furthermore, recent studies found that β-D-glucan ameliorated the progression of immunosuppressive diseases via regulation of the gut microbiota composition [80].

Anti-Inflammation
Inflammation is a comprehensive self-protective response of the body's defense against infection, pathogens, traumas, allergens, and irritants. As anticipated, the antiinflammatory function of β-D-glucan on humans has recently attracted considerable attention [81]. For example, treatment with a water-soluble β-D-glucan from the G. lucidum spores could promote small intestinal crypt epithelial cell proliferation and reduce the levels of pro-inflammatory cytokines, including NO, IL-6, and IL-1β, induced by lipopolysaccharide [6]. Another study confirmed that β-D-glucan from the fruiting bodies of G. lucidum is a favorable potential anti-inflammatory agent, which could suppress not only L -selectinmediated inflammation, but also inhibit the proliferation of mouse spleen lymphocyte and human periphery blood lymphocytes [82]. Mechanistically, β-D-glucan showed potential anti-inflammation by blocking the NF-κB pathway [59] and MAPK pathway [7] (Figure 3).

Antitumor Activity
To date, the high morbidity and mortality of malignancies have become a huge challenge for global public health. Meanwhile, the clinical efficacy of traditional treatment methods (i.e., surgery, radiotherapy, chemotherapy, and immunotherapy) is far from satisfactory, and even the resistance of tumor cells to chemotherapeutic drugs can accelerate tumor progression [83,84]. Clinical studies have confirmed that malignant tumors are characterized by the uncontrollable proliferation, migration, and invasion abilities of cancer cells [85]. Encouragingly, β-D-glucan acts as one of the components in Ganoderma polysaccharides and it has been shown to have an antitumor activity without side effects [48,86], and so it could be used as a promising therapeutic agent for cancer treatment, which is in demand in clinical applications. For example, the administration of 80 mg/kg β-D-glucan from G. formosanum inhibited tumor growth in the lung cancer mice model, and activated the immune response (i.e., enhanced NK cells) and increased the cytokine levels [63]. Another study proved that G. lucidum-derived β-D-glucan showed a cytotoxic activity against leukemic cell proliferation and induced cell apoptosis in vitro, and enhanced pro-apoptotic protein (Bax) expression and reduced anti-apoptotic protein (Bcl-2) expression [33]. Moreover, mushroom β-D-glucan containing (1→3)-β-D-glycosidic in the main chain followed by (1→6)-β-D-glycosidic in the side chain is the most effective structural feature with an antitumor activity [87,88]. For instance, (1→3)-β-D-glucan from the fruiting bodies of G. tsugae inhibited tumor growth [89]. Functionally, administration with β-D-glucan hampered the progression of colon cancer by increasing the number of beneficial intestinal microbiota [90,91] and the fermented product (i.e., SCFAs). Meanwhile, treatment with β-D-glucan from G. lucidum suppressed the malignant biological behavior (i.e., proliferation, invasion, migration, and angiogenesis) of cancer cells via the inhibition of the EGFR/AKT pathway [92] and ERK1/2 pathway [93] (Figure 4).

Antioxidant Activity
Excess reactive oxygen species (ROS) produced during oxidative stress in the human body is a major factor that can cause Alzheimer's disease, diabetes, atherosclerosis, cancer, and other diseases [94]. Clinical studies have confirmed that the reduction of ROS levels by antioxidants may reduce the risk of chronic diseases and age-related health problems [95,96]. Of note, Ganoderma polysaccharides (β-D-glucan) serve as natural antioxidants and have been reported to exhibit a stronger antioxidant activity when scavenging different radicals [25,97]. For example, treatment with β-D-glucan from the fruiting bodies of G. lucidum could reduce ROS levels induced by H 2 O 2 , as well as inhibit SMase activity [30]. Another study confirmed that a novel β-D-glucan obtained from the mycelia of G. capense had a DPPH radical-scavenging ability and an effective concentration 50 value of 3.23 µM [98]. The above results indicate that the potent antioxidant activity of β-D-glucans from Ganoderma species would lay the foundation for their wide application in cosmetic, anti-aging, and pharmaceutical industries. However, the regulatory mechanisms through which β-D-glucan exerts an antioxidant activity are still largely unknown.

Effect of β-D-Glucan on Gut Microbiota
Nowadays, the importance of the gut microbiota in human health and diseases has attracted many researchers' attention [99]. Recently, alterations in the gut microbiota have been highly correlated with the levels of SARS-CoV-2, as well as the severity of patients with COVID-19 [100]. Over the past decade, numerous researches have demonstrated that oral polysaccharides can interact with the gut microbiota to exert nutritional or pharmacological effects [101]. Mechanistically, polysaccharides can regulate the composition of the gut microbiota and modulate the production of gut microbiota metabolites, resulting in the production of a series of metabolites such as SCFAs, secondary bile acids, tryptophan, and indole derivatives. Of note, several in vitro experiments have shown that β-D-glucan treatment facilitated the growth of Lactobacilli and Bifidobacterial [102,103]. Similarly, β-Dglucan isolated from the spores of G. lucidum ameliorated dextran sodium sulfate-induced colitis by increasing the number of SCFA-producing bacteria and reducing pathogens [48]. Sang et al. [104] reported that β-D-glucan extracted from the sporoderm-broken spore of G. lucidum improved high-fat diet-induced obesity, hyperlipidemia, and inflammation through modulation of the gut microbiota composition and enhancing SCFA production. In addition, Ganoderma β-D-glucan inhibited the progression of malignant tumors by modulating the composition of the gut microbiota and increasing SCFA production [91,105]. The above results indicate that β-D-glucan from Ganoderma spp. has a significant impact on changes in the gut microbiota and in turn on human health.

Relationships between the Structure and Bioactivity of β-D-Glucan
The versatile biological activities of β-D-glucan are related to its complex structure features (i.e., M w , monosaccharide compositions, configurations of main and branch chains, and specific glycosidic linkages) [10]. For example, β-(1,3-1,4)-D-glucan or β-(-1,3,6)-Dglucan showed a good antioxidant activity [110]. Furthermore, the triple-helix β-(1,3)glucan possessed anti-tumor effects through activation of the immune-related pathways by inhibiting the malignant behavior of cancer cells [111]. For example, the triple-helix conformation of β-D-glucan from the fruiting bodies of G. lucidum could stimulate lymphocyte proliferation and promote macrophages to form pseudopodia [55]. Moreover, the bioactivities of β-D-glucan were affected by its M w . For example, a larger-M w β-D-glucan (1.07 × 10 5 Da) exhibited a better immunomodulatory activity than low-M w β-D-glucan (1.95 × 10 4 Da) [24], which may be related to its direct recognition by specific receptors on the surface of immune cells [112]. Other studies proved that low-M w β-D-glucan presents various beneficial bioactivities, such as modulation of the gut microbiota [113], as well as anti-inflammation [114], hypoglycemic [115], anti-tumor [116] properties. At present, the relationship between the chemical structures of β-D-glucan and its biological activities has not been fully elucidated, and this mechanism needs to be studied in depth. It has been shown that β-D-glucan with a triple helix conformation and a certain degree of branching can exhibit versatile biological activities [117].

Safeties
Currently, numerous studies have confirmed the beneficial effects of β-D-glucan on human health, but few studies have analyzed the toxicity and safety of β-D-glucan from Ganoderma species. Preclinical studies found that 0.1-10 mg/mL of polysaccharides from the G. lucidum mycelia did not delay the hatching and teratogenic defect on Zebrafish embryos at 24 and 120 h post-fertilization [118]. Chen et al. [119] showed that the administration of β-D-glucan (0, 500, 1000, and 2000 mg/kg/day for 90 days) did not cause any toxicologically significant treatment-related changes in clinical observations, ophthalmic examinations, body weights, body weight gains, feed consumption, and organ weights, as well as no cytotoxic effects on the hematology, serum chemistry parameters, urinalysis, or terminal necropsy, which indicated the safety of β-D-glucan application. A clinical study showed that a total of 88 patients with urinary tract infections were treated with Ganoderma polysaccharides for 12 weeks, and all of the patients had no signs of liver, hematological, or renal toxicities [120]. Until now, a large number of bioactive β-D-glucans from Ganoderma species have been extracted and purified, but the ongoing or completed toxicological studies of these β-D-glucans is just the tip of the iceberg. Therefore, it is necessary to conduct in-depth toxicological studies on β-D-glucan to facilitate confirmation of its efficacy, safety, and potential mechanisms in animal experiments and human trials. Meanwhile, follow-up studies that solve these issues will provide more reliable toxicity and safety data for the application of β-D-glucan from Ganoderma species in food, medicine, and cosmetics.

Applications of β-D-Glucan
As human living standards continue to improve and health awareness grows, consumers are paying more attention to diet and medication for health care. Currently, Ganoderma species that are both edible and medicinal and offer health advantages are now growing in popularity among consumers. Pharmacological and clinical studies have confirmed that polysaccharides extracted from the fruiting bodies, mycelia, spores, and fermentation broths of Ganoderma species have versatile biological activities such as immunomodulation, antitumor, antioxidant, anti-inflammatory, and anti-aging properties [121][122][123], which are widely used in functional foods, multi-purpose drugs, and cosmetics. For example, several healthcare products and foods containing polysaccharides from the fruiting bodies, mycelia, spores, and fermentation broths of Ganoderma species have been developed and produced in markets across the globe, including drinks, healthy wine, jams, and cookies [124][125][126]. Meanwhile, some pharmaceutical commercial products containing Ganoderma polysaccharides are used as dietary supplements for humans in the form of powders, oral liquids, and capsules; in particular, capsule products are used as adjuvant drugs for tumor treatment. Of note, β-D-glucan has been widely used in food and pharmaceutical industries due to its physical properties such as water solubility, viscosity, and gelation. For example, Vanegas-Azuero et al. [127] demonstrated that yogurt containing β-D-glucan showed a high percentage of free amino acids, faster protein hydrolysis, better texture parameters, and high sensory acceptability. A study on healthy children found that the administration of yogurt enriched with β-D-glucan from G. lucidum increased the frequency of peripheral blood total lymphocytes (CD3 + , CD4 + , and CD 8+ T cells), which are critical elements for the body's defense against infectious threats [72]. A randomized controlled trial by Vlassopoulou et al. [128] found that the administration of β-D-glucan with a dosage ranging from 2.5-1000 mg/day for 6.5 months significantly enhanced immune defense, improved allergic symptoms, and decreased comorbid conditions associated with obesity. Another clinical trial confirmed that patients with angina pectoris taking 750 mg/day of β-D-glucan for 90 days had increased superoxide dismutase (SOD) levels, decreased malondialdehyde (MDA) concentrations, and reduced numbers of circulating endothelial cells and endothelial progenitor cells [129]. Moreover, Ganoderma polysaccharides have broad application prospects in animal husbandry and the feed industry as a green and natural feed additive with rich biological functions [130]. New research has shown that β-D-glucan from G. lucidum exhibits whitening effects by reducing tyrosinase activity and melanin synthesis [64]. As a medication delivery system, β-D-glucan has become an important topic in today's research. For example, Takedatsu et al. [131] created a complex form consisting of macrophage-migration inhibitory factor (MIF) and two single schizophyllan (SPG) chains (β-D-glucan) as a new delivery system for antisense oligonucleotides, and treatment with this antisense MIF/SPG complex effectively inhibited MIF production and reduced intestinal inflammation in a dextran sodium sulfate-induced colitis mice model. Collectively, commercial products of Ganoderma β-D-glucan have obtained popularity among humans worldwide for their versatile bioactivities and for being "green and natural", without side effects.

Conclusions and Future Perspectives
Over the past half-century, polysaccharides obtained from natural sources have received increasing attention owing to their diverse health benefits. Ganoderma is rare medicinal fungi mushroom that has been cultured and consumed worldwide for centuries; it has been used as a traditional remedy for many diseases, including cancer, cardiovascular diseases, allergies, and lung deficiency coughs. Polysaccharides are extracted from various Ganoderma species and have the advantages of a low toxicity and broad medicinal value. Because of these properties, Ganoderma polysaccharides have been recognized as functional foods and are considered as a source for the development of drugs, nutritional products, and cosmetics. Importantly, Ganoderma β-D-glucan serves as an effective and desirable polysaccharide to infuse immune health benefits into any kind of food, dietary supplements, pharmaceuticals, and cosmetics.
Based on the fact that Ganoderma species have very different structures and efficacy in different cultured regions, sharing the research results related to Ganoderma species from different regions can enable efficient utilization of Ganoderma polysaccharides and thus broaden its potential market value. Meanwhile, the bioactivities of β-D-glucan extracted from edible and medicinal Ganoderma have received much attention in the biomedical field. However, to further improve the utilization of β-D-glucan in Ganoderma species, future research should focus on the following directions: (1) there is an urgent need to select excellent strains, culture techniques, and/or fermentation strategies to improve the shortage of wild Ganoderma species; (2) using synthetic biology combined with genetic engineering to enhance the productivity and yield of β-D-glucan in Ganoderma species; (3) revealing the relationship between structure and biological activities of β-D-glucan through multi-omics strategies, such as transcriptomics, nutrigenomics, proteomics, and metabolomics; (4) developing food, pharmaceutical, and cosmetic products with β-Dglucan as a functional component and analyzing its safety and toxic side effects; (5) seeking effective physicochemical methods (i.e., ultrasound and microwave) to overcome the special physical properties of Ganoderma β-D-glucan (i.e., high Mw, linkage pattern and high viscosity) may help to obtain small M w of β-D-glucan with good absorption and utilization; (6) More clinical studies are required to investigate the use of food-grade β-D-glucan in humans because the majority of pharmacological studies of β-D-glucan are restricted to in vitro or animal models.
In summary, β-D-glucan extracted from Ganoderma species will have great market potential for use in food, pharmaceuticals, and cosmetics. In addition, overcoming the above-mentioned drawbacks will provide new ideas for the development of natural β-Dglucans into highly efficient and low-toxic novel products.

Data Availability Statement:
The data used and analyzed during the current study are available from the corresponding author on academic request (C.T.). The data are not publicly available to preserve the privacy of the data.

Conflicts of Interest:
The authors declare no conflict of interest.